The activity of yeast NAD-dependent glutamate dehydrogenase (NAD-GDH) is regulated by rapid and reversible interconversion between active and less active enzyme forms. The aim of this project was to purify the active (a) and the less active (b) enzyme forms, and to elucidate the mechanism of reversible interconversion. Both enzyme forms were purified by chromatography on DEAE-cellulose, Cibacron Blue FG3A-agarose and Ultrogel AcA 22. Characterization of the two enzyme forms showed that the b form had different properties compared to the fully active or a form. Analysis of purified NAD-GDHa and NAD-GDHb for alkali-labile phosphate revealed that the a form contained 0.12 plus or minus 0.095 mol of phosphate/mol of enzyme subunit and the b form 1.22 plus or minus 0.09 mol of phosphate/mol of enzyme. The NAD-GDHb was purified after growth of yeast on 32P containing medium, and found to contain radioactivity. Therefore it is concluded that the reversible enzyme interconversion is achieved by phosphorylation/dephosphorylation cycle.